China Animal Husbandry & Veterinary Medicine ›› 2025, Vol. 52 ›› Issue (8): 3918-3926.doi: 10.16431/j.cnki.1671-7236.2025.08.040

• Basic Veterinary Medicine • Previous Articles    

Construction and Application of Salmonella Pullorum Expressing Red Fluorescent Protein

LIU Zijing1,2, SUN Jia3, ZHANG Li1, LUO Chenglong2   

  1. 1. College of Coastal Agricultural Sciences, Guangdong Ocean University, Zhanjiang 524000, China;
    2. State Key Laboratory of Swine and Poultry Breeding Industry, Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China;
    3. College of Animal Science and Technology, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China
  • Received:2024-12-25 Published:2025-08-02

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Abstract: 【Objective】 The purpose of this experiment was to construct Salmonella Pullorum expressing red fluorescent protein (mCherry) to explore its localization and tracing effect in macrophages. 【Method】 The exogenous gene mCherry was inserted into the prokaryotic expression vector pUC19 to obtain the pUC19-mCherry plasmid.Using the genome of Salmonella Pullorum as a template,the endogenous virulence gene virK promoter was amplified by PCR and inserted into the pUC19-mCherry plasmid to drive the expression of mCherry.The recombinant plasmid PvirK-mCherry was constructed and transformed into the Salmonella Pullorum CVCC 533.Single colonies were selected,and fluorescence expression was confirmed using upright fluorescence microscopy.The recombinant fluorescent strain with an multiplicity of infection (MOI) of 200 was co-cultured with RAW264.7 and HD11 cells.The phagocytic effects of the two types of macrophages on the fluorescent strain were detected by DAPI staining and flow cytometry. 【Result】 The double enzyme digestion results of the pUC19-mCherry plasmid showed a 720 bp mCherry gene fragment and a 2 600 bp vector fragment.Red fluorescence was observed after transforming the Escherichia coli DH5α competent cells.The sequencing results of the recombinant plasmid PvirK-mCherry showed that the promoter was successfully inserted into the pUC19-mCherry plasmid.Double enzyme digestion revealed a 990 bp virK gene promoter fragment and a 3 300 bp vector fragment.Fluorescence microscopy of Salmonella Pullorum transformed with PvirK-mCherry plasmid showed red fluorescence.The results of the macrophage phagocytosis test showed that after DAPI staining,fluorescence microscopy revealed that the red fluorescent Salmonella Pullorum strains were aggregated around the blue nuclei.Flow cytometry results showed that the phagocytic efficiency of RAW264.7 cells was 88.06%,and that of HD11 cells was 34.18%. 【Conclusion】 This study successfully constructed the plasmid PvirK-mCherry，after transfection with Salmonella Pullorum,it expressed red fluorescence,and the red fluorescent strain could be phagocytosed by macrophages.This results not only helped to precisely observe the localization of bacteria within host cells,but also provided an economical and effective tool for studying the interaction between Salmonella and host cells,which was of great significance to the health of poultry and the development of breeding industry.

Key words: Salmonella Pullorum; fluorescent bacteria; macrophage; phagocytosis

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